EP3000900B1 - Detection of hpv dna rearrangements - Google Patents

Detection of hpv dna rearrangements Download PDF

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EP3000900B1
EP3000900B1 EP15192470.1A EP15192470A EP3000900B1 EP 3000900 B1 EP3000900 B1 EP 3000900B1 EP 15192470 A EP15192470 A EP 15192470A EP 3000900 B1 EP3000900 B1 EP 3000900B1
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dna
hsv
viral
probes
virus
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EP3000900A1 (en
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Charlotte Mahiet
Fabrice Salvaire
Emmanuel Conseiller
Sébastien BARRADEAU
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Genomic Vision SA
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    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/708—Specific hybridization probes for papilloma
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C12Q1/703—Viruses associated with AIDS
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/705—Specific hybridization probes for herpetoviridae, e.g. herpes simplex, varicella zoster
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention relates to a method for easily, rapidly and accurately detecting rearrangements of a human papilloma virus (HPV) DNA in a viral host cell, tissue or biological fluid obtained from a subject or patient using Molecular Combing and/or DNA stretching in combination with specially designed probes for the HPV DNA sequence.
  • HPV human papilloma virus
  • the disclosure describes a method for monitoring in vitro the effects of anti-viral treatment by following the presence of genomic viral polynucleotides in a virus-infected cell, tissue or a fluid of a patient to be tested as infected by a virus.
  • the invention relates to the detection of a HPV based on DNA hybridization and the use of a kit comprising the probes used to carry out a method of the invention.
  • the disclosure describes a composition comprising said probes.
  • Bensimon, et al., U.S. Patent No. 6,303,296 discloses DNA stretching procedures
  • Lebofsky, et al., WO 2008/028931 discloses detections of genomic sequences
  • Lebofsky, et al., WO 2010/035140 A1 discloses a method for analysis of D4Z4 tandem repeat arrays on human chromosomes 4 and 10 based on stretching of nucleic acid and on Molecular Combing.
  • Molecular Combing is a technique enabling the direct visualization of individual nucleic acid molecules and has been successfully used for the location of myc and certain Human Papillomavirus (HPV) sequences in cervical cancer cell lines as well as for the study of replication kinetics on previously characterized MYC/papillomavirus (HPV18) amplicon and particularly on its origin of replication ( Herrick, et al., Cancer Res. 65(4): 1174-1179 2005 ). Nucleic acid rearrangements and amplifications in tumor cells were correlated with cancer outcome using this technique. Herrick, et al.
  • Epstein-Barr virus DNA obtained from cultured Burkitt's lymphoma cell lines was characterized by FISH on combed DNA, ( Reisinger, et al., Int. J. Cancer 118: 1603-1608 (2006 ). Reisinger's objective was to study the genomic organization of viral genomes and understand and attempt to discriminate between the episomal and integrated forms of EBV in infected eukaryotic cells.
  • DNA from cultured Burkitt's lymphoma cell lines was stretched by Molecular Combing and probed with EBV-specific DNA probes that cover genomic sequences but not the region between the terminal repeat (TR) and the internal repeat 1 (IR1) sequences.
  • Reisinger et al. and Herrick et al. also do not describe methods for detecting rearrangements of viral genomes using probe sets tagged with different haptens that can generate a different color-fluorescence array that will allows the identification of rearrangements within this specific region of the viral DNA.
  • These two references describe the detection of viral DNA in cultured cells but not in tissue or in biological fluids.
  • the standard extraction procedure is modified to allow the extraction of viral DNA from viral particles in order to analyze by Molecular Combing the structure of the viral DNA in the infectious particles.
  • the invention develops a methodology of extraction of high molecular weight DNA from solid tissues such as cornea that is compatible with the analysis by Molecular Combing and that allows the detection of the presence of the infectious forms of HSV DNA in an infected cornea.
  • Stretching nucleic acid in particular viral or genomic DNA provides immobilized nucleic acids in linear and parallel strands and is preferably performed with a controlled stretching factor on an appropriate surface (e.g. surface-treated glass slides). After stretching, it is possible to hybridize sequence-specific probes detectable for example by fluorescence microscopy (Lebofsky, Heilig et al. 2006). Thus, a particular sequence may be directly visualized on a single molecule level. The length of the fluorescent signals and/or their number, and their spacing on the slide provides a direct reading of the size and relative spacing of the probes.
  • this technology can be used to detect the infectious forms of viruses in an infected eukaryotic cell or viral sequences or genome of a derived mutated viral sequence infecting a cell or tissue or present in a fluid of mammals.
  • EP1331277 discloses a method for DNA sequencing and gene identification that allows identification of a single molecule of a target DNA wherein an optically distinguishable material is attached to a DNA recognition unit and the detection is performed in a sequential manner after the hybridization complex between the DNA sequence hybridization unit and the target DNA molecule has been starched to adopt a substantially linear configuration.
  • the viruses that cause human diseases are placed into seven groups based upon the existence of an envelope, the type of the capsid, the nature of the genome (DNA or RNA, double-stranded or single-stranded), size, and target cells (International Committee on Taxonomy of Viruses, http://_www.ictvonline.org; last accessed April 7, 2011).
  • the enveloped DNA viruses include the poxviruses, herpesviruses, and hepadnaviruses.
  • the nonenveloped group includes the adenoviruses, papovaviruses, and parvoviruses.
  • a viral genome is either single-stranded or double-stranded.
  • Single-stranded genomes consist of an unpaired nucleic acid, analogous to one-half of a ladder split down the middle.
  • All RNA viruses are single-stranded except for the double-stranded reoviruses.
  • Double-stranded genomes consist of two complementary paired nucleic acids, analogous to a ladder.
  • All DNA viruses are double-stranded except for the parvoviruses, which have single-stranded DNA.
  • Viral genomes are circular, as in the polyomaviruses, or linear, as in the adenoviruses (Fields, Knipe et al. 2007).
  • the type of nucleic acid is irrelevant to the shape of the genome.
  • RNA viruses the genome is often divided up into separate parts within the virion and is called segmented. Each segment often codes for one protein and they are usually found together in one capsid. Every segment is not required to be in the same virion for the overall virus to be infectious, as demonstrated by the brome mosaic virus (Collier and Oxford 2006).
  • the strands are said to be either positive-sense (called the plus-strand) or negative-sense (called the minus-strand), depending on whether it is complementary to the viral messenger RNA (mRNA) (Fields, Knipe et al. 2007).
  • Positive-sense viral RNA is identical to viral mRNA and thus can be immediately translated by the host cell.
  • Negative-sense viral RNA is complementary to mRNA and thus must be converted to positive-sense RNA by an RNA polymerase before translation.
  • DNA nomenclature is similar to RNA nomenclature, in that the coding strand for the viral mRNA is complementary to it (-), and the non-coding strand is a copy of it (+).
  • retrovirus which includes HIV
  • retrovirus which includes HIV
  • the DNA is then incorporated into the host's genome by an integrase enzyme.
  • the virus genome, or provirus remains in the genome of the infected cell and thereafter replicates as part of the host cell's DNA.
  • Genome size varies greatly amongst different kinds or species of viruses ranging from 1.7 kb for the hepatitis delta virus (HDV) to 309 kb for the Poxvirus.
  • RNA viruses generally have smaller genome sizes than DNA viruses because of a higher error-rate when replicating, and have a maximum upper size limit. Beyond this limit, errors in the genome when replicating render the virus useless or uncompetitive. To compensate for this, RNA viruses often have segmented genomes where the genome is split into smaller molecules, thus reducing the chance of error. In contrast, DNA viruses generally have larger genomes because of the high fidelity of their replication enzymes (Pressing and Reanney 1984).
  • Viruses can undergo genetic changes that occur through different mechanisms.
  • the first process is called genetic drift where individual bases in the DNA or RNA mutate to other bases.
  • Most of these point mutations are "silent" meaning that they do not change the protein that the gene encodes but others can confer evolutionary advantages such as resistance to antiviral drugs.
  • Antigenic shift occurs when there is a major change in the genome of the virus (Pan, Li et al. 2007). This can be a result of recombination or reassortment. When this happens with influenza viruses (Hampson and Mackenzie 2006), pandemics might result.
  • RNA viruses often exist as quasispecies or swarms of viruses of the same species but with slightly different genome nucleoside sequences.
  • segmented genomes confer also evolutionary advantages; different strains of a virus with a segmented genome can shuffle and combine genes and produce progeny viruses that have unique and new characteristics. This is called reassortment or viral sex (Goudsmit 1998).
  • genetic recombination which is the process by which a strand of DNA is broken and then joined to the end of a different DNA molecule can arise. This can occur when viruses infect cells simultaneously and studies of viral evolution have shown that recombination has been rampant in the species studied (Worobey and Holmes 1999). Recombination is common to both RNA and DNA viruses (Umene 1999; Lukashev 2005).
  • the HSV are among the larger viruses with a diameter ranging from about 150 nm to 200 nm. They are enclosed within a loosely fitting envelope that contains glycoprotein spikes (Wildy, Russell et al. 1960). Like other enveloped viruses, herpes viruses are prone to deactivation by organic solvents or detergents and are unstable outside the host's body.
  • the icosahedral capsid houses a core of double-stranded DNA that winds around a proteinaceous spindle in some viruses.
  • the genome sequence of HSV-1 strain 17 first published in 1988, contains 152,261 residues in each strand (Genbank accession number: NC_001806.1; last accessed April 7, 2011) (McGeoch, Dalrymple et al. 1988).
  • the HSV genome is regarded as being composed of two covalently joined segments, the long (L) and short (S) regions (Roizman 1979).
  • the L region consists of a unique sequence (U L ) flanked by a pair of oppositely oriented repeat elements (termed R L , with the terminal and internal copies specifically referred to as TR L and IR L ) (Wadsworth, Jacob et al. 1975).
  • the S region similarly consists of IRs, Us, and TRs.
  • sequences of the R L and Rs elements are distinct, except that there is a 400 base pairs (bp) direct repeat at the genome termini, termed the a sequence, and at least one further copy of this is found at the junction of L and S regions, in the orientation opposite to the terminal copies (Roizman 1979; Davison and Wilkie 1981). Each terminus possesses one overhanging residue, with the 3'-hydroxyl group free (Mocarski and Roizman 1982).
  • Preparations of HSV DNA contain equimolar amounts of four sequence-orientation isomers, in which U L and Us each lie independently in one of two possible orientations with respect to the joint between L and S sequences (Hayward, Jacob et al. 1975; Bataille and Epstein 1997).
  • HSV-1 DNA has a base composition of 68.3% G+C (McGeoch, Dahymple et al. 1988).
  • the G+C content is not constant throughout the genome; the 6.6-kbp R S elements deviate most notably from the mean, with a G+C content of 79.5% (McGeoch, Dolan et al. 1986).
  • the genome of HSV-2 has not been completely sequenced, but it closely resembles that of HSV-1, albeit with a slightly higher G+C content.
  • a viral infection occurs when a virus enters the body through such processes as breathing air contaminated with a virus, eating contaminated food, or by having sexual contact with a person who is infected with a virus.
  • a viral infection may also be caused by an insect bite.
  • the virus invades the inside of the body's cells in order to reproduce.
  • a virus then spreads to other cells and repeats the process. This process of viral infection results in a variety of symptoms that vary in character and severity depending on the type of viral infection and individual factors. Common symptoms of a viral infection include fatigue, flu-like symptoms and fever.
  • viruses that cause a wide variety of viral infections or viral diseases. For example, there are over 200 different viruses that can cause a cold or an upper respiratory infection. Other common viruses include the influenza virus which causes influenza or the flu. The Epstein-Barr virus and the cytomegalovirus cause infectious mononucleosis, the varicella zoster virus (VZV) causes shingles and chickenpox, and HIV causes AIDS.
  • influenza virus which causes influenza or the flu.
  • the Epstein-Barr virus and the cytomegalovirus cause infectious mononucleosis
  • VZV varicella zoster virus
  • HIV causes AIDS.
  • Viruses have different mechanisms by which they produce disease in an organism, which largely depends on the viral species. Mechanisms at the cellular level primarily include cell lysis, the breaking open and subsequent death of the cell. In multicellular organisms, if enough cells die the whole organism will start to suffer the effects. Although viruses cause disruption of healthy homeostasis, resulting in disease, they can also exist relatively harmlessly within an organism.
  • An example would include the ability of the herpes simplex virus (HSV), which causes cold sores, to remain in a dormant state within the human body. This is called latency (Margolis, Elfman et al. 2007) and is a characteristic of the all herpes viruses including the EBV, which causes glandular fever, and the VZV.
  • HSV herpes simplex virus
  • latent viruses might sometimes be beneficial, as the presence of the virus can increase immunity against bacterial pathogens, such as Yersinia pestis (Barton, White et al. 2007).
  • latent chickenpox infections return in later life as the disease called shingles.
  • Some viruses can cause life-long or chronic infections, where the viruses continue to replicate in the body despite the host's defense mechanisms (Bertoletti and Gehring 2007). This is common in hepatitis B and C virus infections. People chronically infected are known as carriers, as they serve as reservoirs of infectious virus. In populations with a high proportion of carriers, the disease is said to be endemic (Rodrigues, Deshmukh et al. 2001). In contrast to acute lytic viral infections this persistence implies compatible interactions with the host organism.
  • HSV-1 infection is one example of a viral infection.
  • HSV was named for the tendency of some herpes infections to produce a creeping rash (Taylor, Brockman et al. 2002). It is the common name for a large family whose members include herpes simplex 1 and 2, the cause of fever blisters and genital infections; VZV, the cause of chickenpox and shingles; cytomegalovirus (CMV), which affects the salivary glands and other viscera; EBV, associated with infection of the lymphoid tissue; and some recently identified viruses (HSV-6, -7, and -8). Prominent features of the family are its tendency toward viral latency and recurrent infections.
  • HSV infections often colloquially called herpes, usually target the mucous membranes.
  • the virus enters cracks or cuts in the membrane surface and then multiplies in basal and epithelial cells in the immediate vicinity. This results in inflammation, edema, cell lysis, and formation of a characteristic thin-walled vesicle.
  • the main diseases of HSV are facial herpes (oral, optic, and pharyngeal), genital herpes, neonatal herpes, and disseminated disease.
  • Herpes labialis otherwise known as fever blisters or cold sores is the most common recurrent HSV-1 infection.
  • Vesicles usually crop up on the muco-cutaneous junction of the lips or on adjacent skin.
  • Herpetic keratitis also called ocular herpes
  • ocular herpes is an infective inflammation of the eye in which a latent virus travels into the ophthalmic rather than the mandibular branch of the trigeminal nerve.
  • Preliminary symptoms are a gritty feeling in the eye, conjunctivitis, sharp pain, and sensitivity to light.
  • Some patients develop characteristic branched or opaque corneal lesions as well. In 25% to 50% of cases, keratitis is recurrent and chronic and can interfere with vision.
  • Type 1 HSV After replication in epithelial tissues, viruses propagate in neurons before becoming latent.
  • Type 1 HSV enters primarily the trigeminal, or fifth cranial, nerve, which has extensive innervations in the oral region while type 2 HSV usually becomes latent in the ganglion of the lumbo-sacral spinal nerve trunk.
  • the virus migrates to the body surface and produces a local skin or membrane lesion, often in the same site as a previous infection. Since the principal location of latent HSV-1 is the trigeminal ganglion, responsible for sensory innervation of the face, most recurrences are located in the eyes or the lips.
  • HSV-1 The seroprevalence of HSV-1 in the general population ranges from 24.5% to 67%, with 30 to 70% of positive subjects experiencing recurrent herpes labialis (Liesegang 2001). Indeed, almost everyone may potentially exhibit an HSV-1 infection event since recent studies using Polymerase Chain Reaction (PCR) on post-mortem tissues showed that nearly 100% of people at least 60 years of age have several HSV-1 genome copies present in the trigeminal ganglion (Wang, Lau et al. 2005). The eye, and particularly the cornea, is the second most frequent location of HSV-1 infection. The prevalence of herpes keratitis is 149/100,000 (Liesegang, Melton et al.
  • HSV-1 ocular infection accounts for about 10% of patients attending corneal clinics (Pramod, Rajendran et al. 1999), and despite the use of topical or oral antiviral agents in the last three decades, herpes simplex keratitis also remains the most frequent cause of infectious corneal opacities in the most developed areas of the world (group 1998), accounting for about 10% of patients undergoing corneal transplantation (Leger, Larroque et al. 2001). Moreover, the natural risk of HSV-1 reactivation in recently grafted cornea is about 25% in the first year following surgery (Lomholt, Baggesen et al.
  • HIV Human immunodeficiency virus
  • AIDS acquired immunodeficiency syndrome
  • Infection with HIV occurs by the transfer of blood, semen, vaginal fluid, pre-ejaculate, or breast milk. Within these bodily fluids, HIV is present as both free virus particles and virus within infected immune cells.
  • the four major routes of transmission are unsafe sex, contaminated needles, breast milk, and transmission from an infected mother to her baby at birth (vertical transmission). Screening of blood products for HIV has largely eliminated transmission through blood transfusions or infected blood products in the developed world.
  • HIV infection in humans is considered pandemic by the World Health Organization (WHO). From its discovery in 1981 to 2006, AIDS killed more than 25 million people. HIV infects about 0.6% of the world's population. In 2005 alone AIDS claimed an estimated 2.4-3.3 million lives of which more than 570,000 were children. Antiretroviral treatment reduces both the mortality and the morbidity of HIV infection.
  • WHO World Health Organization
  • HIV progresses to AIDS at a variable rate affected by viral, host, and environmental factors; most will progress to AIDS within 10 years of HIV infection: some will have progressed much sooner, and some will take much longer (Buchbinder, Katz et al. 1994; 2000).
  • Treatment with anti-retroviral drugs increases the life expectancy of people infected with HIV and even after HIV has progressed to diagnosable AIDS, the average survival time with antiretroviral therapy was estimated to be more than 5 years as of 2005 (Schneider, Gange et al. 2005). Without antiretroviral therapy, someone who has AIDS typically dies within a year (Morgan, Mahe et al. 2002).
  • Viral diseases are diagnosed by a variety of clinical and laboratory methods. These include a clinical assessment of symptoms, viral isolation in cell or animal culture, and serological testing for antibodies to a virus. Making a diagnosis of a viral infection begins with taking a thorough personal and family medical history, including symptoms, and completing a physical examination. Diagnosing some viral infections, such as seasonal influenza, may be made based on a history and physical. Blood tests, such as a complete blood count may be done. A complete blood count measures the numbers of different types of blood cells, including white blood cells (WBCs). Different types of WBCs increase in number in characteristic ways during an infectious process, such as viral infection. A culture test may also be performed.
  • WBCs white blood cells
  • This test requires taking a small sample from the body area that is suspected to be infected with a virus and grows the sample in a lab to determine the type of microorganism causing illness.
  • Common samples tested with a culture include those from the throat, blood, and sputum from the lungs.
  • Diagnostic tests may also include a lumbar puncture, also called a spinal tap, which involves withdrawing a small sample of cerebrospinal fluid (CSF) from the spine with a needle.
  • CSF cerebrospinal fluid
  • the sample of CSF is tested for white blood cells and other indications of viral infection that may be in the spine or brain, such as viral meningitis.
  • X-rays may be performed to assist in the diagnosis of some viral infections. This may include taking a chest X-ray for suspected cases of viral pneumonia. Additional tests may be performed in order to rule out or confirm other diseases that may accompany viral infection or cause similar symptoms, such as a secondary bacterial infection.
  • HSV infections are diagnosed by a variety of clinical and laboratory methods, including a clinical assessment of symptoms, isolation in cell or animal culture, and serological testing for antibodies to HSV.
  • Small, painful, vesiculating lesions on the mucous membranes of the mouth or genitalia, lymphadenopathy, and exudate are typical diagnostic symptoms of herpes simplex. Further diagnostic support is available by examining scrapings from the base of such lesions stained with Giemsa, Wright (also called Tzanck preparation), or Papanicolaou (Pap) methods.
  • Giemsa also called Tzanck preparation
  • Papanicolaou Papanicolaou
  • Acyclovir is the most effective therapy developed to date that is nontoxic and highly specific to HSV. Famciclovir and valacyclovir are alternate drugs. Topical medications applied to genital and oral lesions cut the length of infection and reduce viral shedding. Systemic therapy is available for more serious complications such as herpes keratitis and disseminated herpes. New evidence indicates that a daily dose of oral acyclovir taken for a period of 6 months to one year can be effective in preventing recurrent genital herpes.
  • Over-the-counter cold sore medications containing menthol, camphor, and local anesthetics lessen pain and may protect against secondary bacterial infections, but they probably do not affect the progress of the viral infection.
  • Some protection in suppressing cold sores can be obtained from the amino acid lysine, taken orally in the earliest phases of recurrence.
  • current treatments do not reduce the load of the DNA matrix, and thus are unable to reduce the risk of further viral reactivation.
  • an ultimate weapon against HSV-1 infection should be durably present in the cells, avoid questions of sensitivity and, if possible, reduce the load of viral genomes.
  • New antiviral treatments with the aim to eliminate the virus DNA by the action of endonucleases are in development.
  • Virus DNA detection of HSV in cornea have been performed from tear fluid (Fukuda, Deai et al. 2008), corneal scrapings (E1-Aa1, E1 Sayed et al. 2006) and corneal explants button from healthy and diseased patients (Crouse, Pflugfelder et al. 1990) using PCR-based methods (Llorente, Hidalgo et al. 1998; Gonzalez-Villasenor 1999; Kessler, Muhlbauer et al. 2000; O'Neill, Wyatt et al. 2003; Kimura, Ihira et al. 2005; Namvar, Olofsson et al.
  • HSV is one type of viral disease for which a large spectrum of technologies for the diagnosis of HSV infection has been disclosed in various patents and publications as for example in EP0139416 , EP0263025 , WO0202131 , WO2004036185 , and in Matsumoto, Yamada et al. 1992, Kotronias and Kapranos 1998, Kessler, Muhlbauer et al. 2000, Namvar, Olofsson et al. 2005, Susloparov et al. 2006, Sugita, Shimizu et al. 2008, and Yu, Shi et al. 2009.
  • HIV-1 load in blood plasma is a major laboratory marker widely used in clinical practice. Higher virus loads are directly linked to more rapid progression to AIDS in HIV-1-infected individuals.
  • HAART highly active antiretroviral therapy
  • HAART cannot eradicate the virus, mainly due to the persistence of various viral reservoirs including latently infected resting CD4 + cells (Hermankova, Siliciano et al. 2003; Siliciano, Kajdas et al. 2003). Recent studies demonstrated that both virus replication and evolution do continue in some patients even when HIV-1 RNA in plasma is undetectable and therapy is otherwise considered to be successful (Frenkel, Wang et al. 2003; Havlir, Strain et al. 2003; Ramratnam, Ribeiro et al. 2004; Chun, Nickle et al. 2005; Tobin, Learn et al. 2005).
  • HAART failure as a result of development of drug-resistant HIV-1 strains is a common problem (del Rio 2006).
  • special attention should be given to characterizing HIV-1 residual replication by studying its molecular markers in peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • the amounts of cell-associated HIV-1 RNA and proviral DNA should be quantified.
  • the amounts of proviral DNA may reflect the size of the pool of latently infected cells.
  • systematic studies of the relationships between the cellular HIV-1 RNA/DNA levels and therapy outcome are hindered by the extremely low copy numbers of HIV-1 RNA/DNA in PBMC under HAART. Therefore, development of highly sensitive methods for quantification of cellular forms of HIV-1 RNA/DNA is essential.
  • RT-PCR Real-time reverse transcription-PCR
  • PCR technique which allows the enzymatic amplification of minute quantities of DNA often undetectable by other methods, has been widely used for detection of part of genome of several viral agents such as HSV (Rowley, Whitley et al. 1990), human immunodeficiency virus (HIV) (Ou, Kwok et al. 1988) and human papilloma viruses (HPV) (Shibata, Arnheim et al. 1988).
  • PCR does not allow one to confirm the integrity of the complete genome detected and consequently cannot be a standard of characterization of complete infectious viral genome found in a tested sample. PCR thus also lacks the specificity required for testing the efficiency of an antiviral treatment against infectious virus particles because its results do not indicate whether the viral polynucleotides detected are infectious. Serological methods do not directly detect infectious viral polynucleotides, such as chromosomally-integrated viral genomic DNA, and require that a subject mount an immune response to a virus prior to detection or that a sufficient amount of viral antigen be present in a sample.
  • Tumor viruses come in a variety of forms: viruses with a DNA genome, such as HPV (cervical cancer), Hepatitis B virus (liver cancer), and EBV (a type of lymphoma), and viruses with an RNA genome, like the Hepatitis C virus (HCV) can cause cancers, as can retroviruses having both DNA and RNA genomes (Human T-lymphotropic virus and hepatitis B virus, which normally replicates as a mixed double and single-stranded DNA virus but also has a retroviral replication component).
  • HPV cervical cancer
  • Hepatitis B virus liver cancer
  • EBV a type of lymphoma
  • RNA genome like the Hepatitis C virus (HCV) can cause cancers, as can retroviruses having both DNA and RNA genomes (Human T-lymphotropic virus and hepatitis B virus, which normally replicates as a mixed double and single-stranded DNA virus but also has a retroviral replication component).
  • a direct oncogenic viral mechanism involves either insertion of additional viral oncogenic genes into the host cell (acutely-transforming virus) or to enhance already existing oncogenic genes (proto-oncogenes) in the genome (slowly-transforming virus) (for review, (Parsonnet 1999)).
  • acutely-transforming viruses the viral particles carry a gene that encodes for an overactive oncogene called viral-oncogene (v-onc) and the infected cell is transformed as soon as v-onc is expressed.
  • v-onc viral-oncogene
  • the virus genome is inserted, especially as viral genome insertion is obligatory part of retroviruses, near a proto-oncogene in the host genome.
  • the viral promoter or other transcription regulation elements cause over-expression of that proto-oncogene, which, in turn, induces uncontrolled cellular proliferation. Because viral genome insertion is not specific to proto-oncogenes and the chance of insertion near that proto-oncogene is low, slowly-transforming viruses have very long tumor latency compared to acutely-transforming virus, which already carries the viral-oncogene.
  • Some viruses are tumorigenic when they infect a cell and persist as circular episomes or plasmids, replicating separately from host cell DNA (Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus). Indirect viral oncogenicity also exists and involves chronic nonspecific inflammation occurring over decades of infection, as is the case for HCV-induced liver cancer. These two mechanisms differ in their biology and epidemiology: direct tumor viruses must have at least one virus copy in every tumor cell expressing at least one protein or RNA that is causing the cell to become cancerous. Foreign virus antigens are expressed in these tumors, consequently persons who are immunosuppressed such as AIDS or transplant patients are at higher risk for these types of cancers.
  • Gene therapy involves the insertion of genes into an individual's cells and tissues to treat diseases, such as hereditary diseases where deleterious mutant alleles of a gene are replaced with functional ones. Although the technology is still in its infancy, it has been used with some success.
  • Viral vectors are a tool commonly used by scientific to deliver such genetic material into tissues (gene therapy) or cells (cellular therapy). These viral vectors are mainly derived from lentiviruses, retroviruses, adenoviruses or herpes viruses (for review, (Thomas, Ehrhardt et al. 2003)).
  • the genetic material in lentiviruses or retroviruses is in the form of RNA molecules, while the genetic material of their hosts is in the form of DNA.
  • the genome of these classes of viruses can be modified by inserting a gene sequence of interest to be transferred in the infected tissue or cells.
  • the recombinant viruses will introduce its RNA together with some enzymes, namely reverse transcriptase and integrase, into the cell.
  • This RNA molecule from the recombinant virus must produce a DNA copy from its RNA molecule before it can be integrated into the genetic material of the host cell. After this DNA copy is produced and is free in the nucleus of the host cell, it must be incorporated into the genome of the host cell. That is, it must be inserted into the chromosomes in the cell by another enzyme carried in the virus called integrase. If this host cell divides later, its descendants will all contain the new genes.
  • the integrase enzyme can insert the genetic material of the virus into any arbitrary position in the genome of the host; it randomly shoves the genetic material into a chromosome. If genetic material happens to be inserted in the middle of one of the original genes of the host cell, this gene will be disrupted (insertional mutagenesis). If the gene happens to be one regulating cell division, uncontrolled cell division (i.e., cancer) can occur.
  • This problem has recently begun to be addressed by utilizing single-chain homing endonucleases (Grizot, Smith et al. 2009) or by including certain sequences such as the locus control region to direct the site of integration to specific chromosomal sites (Zhou, Zhao et al. 2007).
  • Adenoviruses are viruses that carry their genetic material in the form of double-stranded DNA. They cause respiratory, intestinal, and eye infections in humans (especially the common cold). When these viruses infect a host cell they introduce their DNA molecule into the host. The genetic material of the adenoviruses is not incorporated into the host cell's genetic material. The DNA molecule is left free in the nucleus of the host cell and the instructions in this extra DNA molecule are transcribed just like any other gene. The only difference is that these extra genes are not replicated when the cell is about to undergo cell division so the descendants of that cell will not have the extra gene.
  • Adeno-associated viruses (AAV) from the parvovirus family are small viruses with a genome of single stranded DNA.
  • the wild type AAV can insert genetic material at a specific site on chromosome 19 with near 100% certainty (Huser and Heilbronn 2003). But the recombinant AAV, which does not contain any viral genes and only the therapeutic gene, does not integrate into the genome. As a result, treatment with either adenoviral vector or AAV will require readministration in a growing cell population although the absence of integration into the host cell's genome should prevent the development of cancer (Douglas 2007).
  • Herpes viruses are currently used as gene transfer vectors due to their specific advantages over other viral vectors.
  • HSV derived vectors are the very high transgenic capacity of the virus particle allowing to carry long sequences of foreign DNA, the genetic complexity of the virus genome, allowing to generate many different types of attenuated vectors possessing oncolytic activity, and the ability of HSV vectors to invade and establish lifelong non-toxic latent infections in neurons from sensory ganglia from where transgenes can be strongly and long-term expressed.
  • Three different classes of vectors can be derived from HSV: replication-competent attenuated vectors, replication-incompetent recombinant vectors and defective helper-dependent vectors known as amplicons.
  • Replication-defective HSV vectors are made by the deletion of one or more immediate-early genes, e.g. ICP4, which is then provided in trans by a complementing cell line.
  • Oncolytic HSV vectors are promising therapeutic agents for cancer. Such HSV based vectors have been tested in glioma, melanoma and ovarian cancer patients.
  • the inventors have surprisingly found that DNA obtained directly from biological samples from a virus-infected subject can be evaluated by Molecular Combing procedures and active, infectious viral DNA detected in these samples. These methods overcome many of the problems associated with existing diagnostic methods for virus infection and provide a convenient, rapid and accurate method for detection and monitoring of infectious viral polynucleotides in a virus-infected subject or patient.
  • the invention provides a reliable, simple, fast, and inexpensive way to detect rearrangements of a human papilloma virus (HPV) DNA, in a sample from a subject or patient using a Molecular Combing and/or DNA stretching techniques in combination with specific probes which are able to bind with at least 5%, 10%, 25%, 50%, 75%, especially sets of probes that bind to 80-100% of the HPV genome.
  • the method of the invention is carried out in vitro.
  • Molecular Combing is a powerful technique (i) to detect and quantify viral DNA or viral origin-DNA in infected mammalian cells, tissues or biological fluids, such as infectious herpes simplex virus DNA in infected cornea, (ii) follow viral replication and viral genomic rearrangements that occur in the infected mammalian cells tissues or biological fluids, and (iii) identify infectious virus polynucleotides or infectious virus polynucleotides present in infected cells, tissues or biological fluids.
  • the Molecular Combing techniques of the invention can be performed simultaneously and do not require multiple time consuming and expensive procedures.
  • the methods of the invention are easily applicable as diagnostic tools for detecting viral contamination or infection in cells or tissues, useful for determining the efficacy of antiviral treatments by quantifying or otherwise evaluating the effects or efficacies of such treatments on the quantity of infectious virus or infectious viral polynucleotides or viral replication in infected mammalian cells or tissues.
  • the inventors have found that Molecular Combing overcomes many of the problems associated with past methods of detecting or diagnosing a viral infection, including PCR-based methods, and offers an easy and rapid way to do so. Specific embodiments of the invention include the following.
  • a method for detecting rearrangements of a HPV DNA in a biological sample comprising extracting a polynucleotide from said sample; Molecular Combing said HPV DNA to form a stretched polynucleotide; contacting the stretched polynucleotide with a set of labelled probes specific for HPV DNA that recognize a HPV sequence; detecting hybridization of the probes to the combed HPV DNA, thereby detecting rearrangements of the HPV DNA.
  • the polynucleotide may be infectious genomic viral DNA or infectious non-genomic viral DNA, an oncogene or a protooncogene and may be integrated into a chromosome of a subject or may be episomal or transgenic DNA.
  • Detection of an entire viral genome or entire infectious viral DNA is importantly correlated with infection.
  • HSV causative agents of herpetic keratitis it is essential to be able to show the presence of complete infectious genomes in the cells which can then be partially or totally generated in the infected cells.
  • the biological sample is generally obtained directly from a subject or patient and may constitute tissue, a cell sample, or blood, CSF, or synovial fluid sample or other fluid biological sample.
  • a DNA molecule or fiber for stretching or for Molecular Combing may be extracted from the biological sample.
  • a sample is obtained from a living or non-living subject.
  • Subjects include animals susceptible to viral diseases including humans, non-human mammals, such as cattle, bovines, sheep, goats, horses, pigs, dogs, cats and non-human primates; birds, such as chicken, turkey, duck, goose, ostrich, emu, or other birds; reptiles, amphibians and other animals.
  • a stretched or combed DNA molecular is contacted with a set of labelled probes specific for HPV DNA that recognize a HPV sequence.
  • a set of probes covering 80-100% or any intermediate subrange or value of the HPV genome are employed.
  • the HPV DNA may be one that is single or double stranded.
  • representative DNA viruses include herpes virus, such as Herpes Simplex Virus (HSV) types 1 and 2, papillomaviruses, and hepatitis viruses, such as hepatitis B virus.
  • Probes that bind to retroviruses, such as HIV, preferably a retrovirus integrated as a provirus into a host chromosome are also selected in a similar manner and preferably a set of probes covering 80-100% of the proviral genome are employed.
  • the probes may correspond to at least 5% and preferably 80-100% of the genome of a HPV strain already known to be infectious or may be designed to encompass the combination of genes known to be essential for replication, reproduction, or pathogenicity of a particular kind of virus.
  • Said probes can correspond to HPV16 (NC_001526.2)(SEQ ID NO: 20), HPV18 (XO5015.1)(SEQ ID NO: 21), HPV31 (J04353.1)(SEQ ID NO: 22), HPV33 (M12732.1)(SEQ ID NO: 23) and HPV45 (X74479.1)(SEQ ID NO: 24).
  • the probe set may comprise two, three or more different subsets of probes tagged with different labels.
  • probes are used to determine the configurations of a genomic or infectious HPV DNA or to identify different portions or segments of a HPV genome or infectious HPV DNA molecule.
  • the disclosure also describes a method for detecting or identifying an infectious or genomic viral polynucleotide sequence in a mammalian cell, tissue or biological fluid comprising using Molecular Combing to detect the presence or the quantity of infectious viral polynucleotide or genomic viral polynucleotide in a cell, tissue or biological fluid.
  • the disclosure describes a method for quantifying an infectious or genomic viral polynucleotide sequence comprising using Molecular Combing to detect quantity of infectious viral polynucleotide or genomic viral polynucleotide in a cell, tissue or biological fluid compared to an uninfected control sample or an otherwise similar sample obtained at a different point in time.
  • methods for detecting the presence of a virus or detecting replication of a virus in a biological sample are also contemplated, as well as methods for longitudinally following virus replication or the rearrangement of a viral genome or infectious viral DNA especially in mammalian cells, tissue or biological fluid.
  • Such methods may be performed by Molecular Combing DNA obtained from appropriate samples or samples taken over a specific time period for the presence of latent or replicating viral DNA or rearranged viral DNA in a cell or tissue.
  • the disclosure also describes a method for evaluating the efficacy of anti-viral treatment using Molecular Combing to detect the presence, arrangement or quantity of infectious or genomic viral DNA in a sample obtained from a subject, treating the subject with an anti-viral agent or performing other anti-viral therapy, and monitoring or re-evaluating the presence, arrangement or quantity of infectious or genomic viral DNA after treatment using Molecular Combing techniques of described herein.
  • kits comprising reagents, comprising 0.1% SDS for the lysis of viral particles in low-melting agarose plugs and one or more probes that bind to an infectious viral polynucleotide for detecting or identifying genomic viral HPV DNA, in a combed DNA molecule.
  • the kits will contain a set of probes for detecting or identifying genomic viral DNA in a combed DNA molecule covering at least 5% of a HPV genome, especially 80, 85, 90, 95, 99 or 100%.
  • the minimal length of the probe or set probe is 1 kb.
  • the kit may further contain software for detecting or classifying the infectious sequences and the kit may additionally comprise instructions for detecting a HPV genome according to the methods of the invention.
  • the invention enables a rapid, specific and sensitive detection of rearrangements of a HPV DNA in a sample that avoids the significant constraints imposed by amplification methods like PCR or serological tests such as ELISA.
  • the Molecular Combing techniques of the invention permit the successful detection of complete viral genomes or infectious or virulent portions of viral polynucleotide sequences leading to improved diagnosis of acute or latent viral infections.
  • the method disclosed herein enables to detect the type of HSV and the structure of its genome reliably, in a time- and cost-effective fashion, and with none of the constraints of manipulating radioactivity. Moreover, the method disclosed herein enables to follow the presence of the viral infection after antiviral treatment, whatever the type of treatment considered.
  • the disclosure describes a method for detecting in vitro the presence of a genome of DNA viruses in infected cells of eukaryotic cells, in particular the detection of the HSV genome.
  • Said method comprises a hybridization step of nucleic acid representative of given virus with at least a probe or a set of probes which is (are) specific for HSV DNA.
  • the inventors modified the standard extraction protocol to isolate viral genomic DNA from viral particles. Generally, 0.1% SDS is used for the lysis of viral particles in the low-melting agarose plugs instead of 0.1% sarkosyl in the standard protocol. A methodology was also developed to extract genomic DNA from cornea to investigate the presence of HSV DNA in infected cornea for purposes of diagnosis.
  • the present invention relates to a method for detecting rearrangements of a HPV DNA contained on nucleic acid of infected cells, tissue or biological fluid.
  • Said method comprises a hybridization step of nucleic acid representative of said HPV DNA with a set of labelled probes that cover the entire said HPV DNA and that permit to identify rearrangements within the nucleic acid representative of said HPV DNA.
  • nucleic acid and in particular "nucleic acid representative of viral DNA” as used herein designates one or several molecules of any type of nucleic acid capable of being attached to and stretched on a support as defined herein, and more particularly stretched by using Molecular Combing technology; nucleic acid molecules include DNA (in particular genomic DNA, especially viral DNA, or cDNA) and RNA (in particular mRNA). A nucleic acid molecule can be single-stranded or double-stranded.
  • nucleic acid representative of said HPV DNA means that said nucleic acid contains the totality of the genetic information or the essential information with respect to the purpose of the invention, which is present on said HPV DNA.
  • genomic HPV DNA such as that integrated into a host chromosome and which can produce infectious virus, infectious HPV DNA which may lack certain genomic elements but can be infectious when expressed in particular host cells, and viral genes which exert a pathogenic effect on host cells, such as viral oncogenes.
  • a proto-oncogene includes those normal genes, which when altered by mutation can convert into oncogenes that causes a cell to grow or divide in an unregulated manner.
  • Proto-oncogenes have diverse cellular functions, some provide signals for cell division, and others may play roles in apoptosis.
  • Functional and structural characteristics of oncogenes, including their nucleic acid sequences, are well-known in the art and are described in Human Cancer Viruses: Principle of Transformation and Pathogenesis by J. Nicolas, et al., Karger Publishers (2010), ISBN3805585764, 9783805585767 (244 pages ).
  • an oncogene encompasses a defective version of a proto-oncogene.
  • a single copy of an oncogene can cause uncontrolled cell growth.
  • Representative oncogenes include ras, myc, src, Her-2/neu, hTERT, and Bcl-2.
  • Functional and structural characteristics of oncogenes, including their nucleic acid sequences, are well-known in the art and are described in Oncogene: Gene, Mutation, Tumor, Apoptosis, Gene Expression, Protein, Cell Growth, Cellular Differentiation, by L. M. Surhone, et al., Betascript Publishers (2010), ISBN6130361599, 9786130361594 (172 pages ).
  • oncogenes oncogenes
  • the process of activation of proto-oncogenes to oncogenes can include viral transduction or viral integration, point mutations, insertion mutations, gene amplification, chromosomal translocation and/or protein-protein interactions.
  • Viruses that can induce activation of proto-oncogenes include HBV and HCV (hepatocellular carcinoma), HTLV (leukaemia), HPV (cervical, anal and penile cancer), HSV-8 (Kaposi's sarcoma), Merkel cell polyomavirus (Merkel cell carcinoma) and, EBV (Burkitt's lymphoma, Hodgkin's lymphoma, post-transplantation lymphoproliferative disease and Nasopharyngeal carcinoma).
  • the nucleic acid sample used for stretching may be genomic DNA, in particular total genomic DNA or more preferably chromosomal genomic DNA (nuclear genomic DNA) of infected cells or tissues, and/or fragments thereof.
  • the term "nucleic acid” is in particular used herein to designate a nucleic acid representative of one or several chromosome(s) and/or of one or several fragment(s) of chromosomes.
  • Said fragments can be of any size, the longest molecules reaching several megabases.
  • Said fragment are generally comprised between 10 and 2000 kb, more preferably between 20 and 500 kb and are in average of about 300 kb.
  • the nucleic acid sample used in the method of the invention is obtained from a biological fluid or from a tissue of biological origin, said sample or tissue being isolated for example from a human, a non human mammal or a bird.
  • a probe is a polynucleotide, a nucleic acid/polypeptide hybrid or a polypeptide, which has the capacity to hybridize to nucleic acid representative of virus DNA as defined herein, in particular to RNA and DNA.
  • This term encompasses RNA (in particular mRNA) and DNA (in particular viral cDNA or viral genomic DNA) molecules, peptide nuclear acid (PNA), and protein domains.
  • Said polynucleotide or nucleic acid hybrid generally comprises or consists of at least 100, 300, 500 nucleotides, preferably at least 700, 800 or 900 nucleotides, and more preferably at least 1, 2, 3, 4 or 5 kb.
  • probes of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 kb or more than 15 kb, in particular 30, 50, 100 or 150 kb can be used.
  • Such a probe or a set of probes may correspond or cover 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 98, 99 or 100% of a viral genome, especially that of an infectious virus.
  • a nucleic acid probe may be a single or double-stranded polynucleotide or a modified polynucleotide.
  • a set of continuous set of probes will cover a particular section of the genome or the entire genome or overlap each other with respect to this section.
  • a suitable probe may be based on a viral polynucleotide sequence, such as those disclosed herein, and may be identified or synthesized by any appropriate method. Probes may be labelled or tagged radioactivity, fluorescently or by other means known in the art.
  • a polypeptide probe generally specifically binds to a sequence of at least 6 nucleotides, and more preferably at least 10, 15, or 20 nucleotides.
  • sequence of a probe when the probe is a polypeptide, should be understood as the sequence to which said polypeptide specifically binds.
  • a portion of a particular region it is meant herein consecutive nucleotides of the sequence of said particular region.
  • a portion according to the invention can comprise or consist of at least 15 or 20 consecutive nucleotides, preferably at least 100, 200, 300, 500 or 700 consecutive nucleotides, and more preferably at least 1, 2, 3, 4 or 5 consecutive kb of said particular region.
  • a portion can comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 consecutive kb of said particular region.
  • the probe used or at least one of the probes used is a nucleotide variant of the probe showing a complementary sequence of 100% to a portion of one strand of the target nucleic acid.
  • the sequence of said variant can have at least 70, 80, 85, 90 or 95% complementarity to the sequence of a portion of one strand of the target nucleic acid.
  • Said variant can in particular differ from the probe which is 100% identical or complementary by 1 to 20, preferably by 1 to 10, nucleotide deletion(s), insertion(s) and/or more preferably substitution(s), in particular by, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotide deletion(s), insertion(s) and/or more preferably substitution(s) in the original nucleotide sequence.
  • the variant keeps the capacity to hybridize, in particular to specifically hybridize, to the sequence of the nucleic acid target, similarly to the probe that is 100% identical or 100% complementary to a sequence of the nucleic acid target (in particular in the hybridization conditions defined herein).
  • complementary sequences in the context of the invention means “complementary” and “reverse” or “inverse” sequences, i.e. the sequence of a DNA strand that would bind by Watson-Crick interaction to a DNA strand with the said sequence.
  • the probes or one or several probes used to carry out the invention may be labelled with one or several hapten(s) (for example biotin and digoxygenin) and revealed with specific antibodies directed against these haptens.
  • haptens for example biotin and digoxygenin
  • Use of different haptens for a given probe or set of probe will allow to detect rearrangements within a given viral DNA.
  • Said probes can be labelled as defined herein and as described in patent application WO 2008/028931 .
  • a set of probes as used herein comprises of at least two probes.
  • said set of probes can consist of 2 to 20 probes (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 probes).
  • the number of probes in a set does usually not exceed 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 probes depending on the sensitivity that is required and the length of the probe; a set of probes preferably consists of 2, 3, 4, 5, 6, 7, 8, 9 or 10 probes at the most.
  • the probes or the set of probes of the invention not only allow the detection of the entire whole genomic HPV DNA but also to identify rearrangements that can occur within the genomic HPV DNA.
  • the probe sets used in the Example 1 for HSV detection allow the detection of the different HSV genome isomers that are generated by homologous recombination between the inversed repeated sequences that surround the unique regions U L and Us.
  • each probes set (H1 to H6) for the detection of HSV are composed of several probes (3 to 19 different fragments) that are labeled with different two different haptens (digoxygenin for the fragments consisting of H1, H3 and H5 probes; biotin for the fragments consisting of H2, H4 and H6 probes).
  • haptens of one or of several fragments by other haptens permits the generation of a different color-fluorescence array that allows the identification of rearrangements within this specific region of the viral DNA.
  • Molecular Combing can also be a useful tool for an early diagnosis of patients susceptible of developing a cancer caused by a viral infection, for example, those at risk of conversion of a proto-oncogene into an oncogene. Indeed, Molecular Combing can discriminate between integrated and episomal viral genome. In the case where the viral DNA is integrated, by using a specific set of probes Molecular Combing will allow to determine whether the integrated viral DNA contains a complete viral oncogene or whether it is integrated in a close proximity of a cellular proto-oncogene.
  • the invention may also be used in conjunction with gene therapy.
  • Molecular Combing provides a powerful tool for an evaluation of efficacy and safety of the viral vector-based gene therapy. Indeed, Molecular Combing can discriminate between integrated and episomal transgene.
  • the transgene is integrated (lentiviruses and retrovirus based vectors)
  • Molecular Combing will allow to determine whether the integrated viral DNA is integrated in a gene (insertional mutagenesis) or in a close proximity of a cellular proto-oncogene.
  • transgene is episomal (Adenoviral vector, AAV or HSV vector); Molecular Combing will be useful for the quantification of the transgene in the growing cell population and may help to define the right time for readministration of the transgene.
  • a method for detecting rearrangements of a HPV DNA in a biological sample comprising: extracting a polynucleotide from said sample, Molecular Combing said HPV DNA to form a stretched polynucleotide, contacting said stretched polynucleotide with a set of labelled probes specific for HPV DNA that recognize a HPV sequence, detecting hybridization of the probes to the combed HPV DNA; thereby detecting rearrangements of the HPV DNA.
  • This method is performed using a biological sample that is a tissue or cell sample obtained from a subject, for example, a blood, plasma, serum, CSF, synovial fluid sample or some other kind of biological fluid from the subject.
  • a polynucleotide used in this method may be extracted from the tissue or cell sample or from components of a biological fluid obtained from the subject. Generally, the sample will be obtained from a living subject, but samples may also be obtained from deceased subjects. Samples can be obtained from humans or other mammals such as cattle, bovines, sheep, goats, horses, pigs, dogs, cats and non-human primates, or from other animals such as avian species such as a chicken, turkey, duck, goose, ostrich, emu, or other bird. The method can be practiced with polynucleotides which are DNAs which may also contain or comprise infectious or non-infectious genomic viral DNA or non-infectious non-genomic viral polynucleotides such as DNA or RNA.
  • the polynucleotide detected or analyzed by this method may be integrated into the DNA of the subject or can be in episomal form.
  • the polynucleotide to be detected can be contacted with one or more probes that bind to a DNA virus, including both single-stranded and double-stranded DNA viruses.
  • the polynucleotide may be contacted with one or more probes that bind to a herpes virus, such as herpes simplex virus (HSV).
  • HSV herpes simplex virus
  • other probes may be used which bind to other viruses like papilloma virus, hepatitis B virus, or retroviruses like HIV.
  • the method will use a set of probes that bind to at least 80%, 90%, 95%, or 99% of the genomic or infectious DNA of a virus.
  • the set of probes used in the method of the invention will comprise at least two subsets of probes that are tagged with different labels, for example, to permit the identification of different portions or segments of a viral genome to which the different probes bind or to permit the identification of rearrangements in a viral genome.
  • the disclosure also describes:
  • a method for detecting, identifying or visualizing an infectious or genomic viral polynucleotide sequence in a mammalian cell, tissue or biological fluid comprising using Molecular Combing to detect the presence or the quantity of infectious viral polynucleotide or genomic viral polynucleotide in a cell, tissue or biological fluid.
  • a method for quantifying an infectious or genomic viral polynucleotide sequence comprising using Molecular Combing to detect quantity of infectious viral polynucleotide or genomic viral polynucleotide in a cell, tissue or biological fluid compared to an uninfected control sample or an otherwise similar sample obtained at a different point in time or from a different source or clinical sample.
  • a method for detecting or following viral presence or replication or viral genomic rearrangements in a mammalian cell comprising Molecular Combing for the presence of latent or replicating viral DNA or rearranged viral DNA in a cell, tissue or biological fluid.
  • a method for evaluating the efficacy of anti-viral treatment comprising detecting using Molecular Combing the presence, arrangement or quantity of infectious or genomic viral DNA in a sample obtained from a subject, treating said subject with an anti-viral agent, and re-evaluating using Molecular Combing the presence, arrangement or quantity of infectious viral or genomic viral DNA in said subject.
  • the invention relates to a set of probes covering 5-100% of the HPV genome.
  • the invention also relates to the use in the method of the invention of a kit comprising reagents, comprising 0.1% SDS for the lysis of viral particles in low-melting agarose plugs and one or more probes that bind to an infectious viral polynucleotide for detecting or identifying genomic viral HPV DNA in a combed DNA molecule.
  • the kit may further compre software for detecting or classifying the infectious sequences.
  • the disclosure describes the following biological materials which have been deposited under the terms of the Budapest Treaty at the CNCM, Institut Pasteur 25 Rue du Do Budapest Treaty at the CNCM, Institut Pasteur 25 Rue du Do Budapest Treaty at the CNCM, Institut Pasteur 25 Rue du Do Budapest Treaty at the CNCM, Institut Pasteur 25 Rue du Do Budapest Treaty at the CNCM, Institut Pasteur 25 Rue du Do Budapest Roux, F-75724 Paris Cedex 15 on April 20, 2010: HSV-B4 (CNCM I-4298), HSV-B19 (CNCM I-4299), HSV-Sc54 (CNCM I-4300), and HSV-P4 (CNCM I-4301). Each biological material contains a polynucleotide fragment corresponding to a probe for the detection of HSV. The disclosure describes each of these polynucleotide fragments and to any of their combinations.
  • HSV-1 DNA was extracted from viral particles by standard phenol:chloroform extraction (Ben-Zeev, Weinberg et al. 1974) or by a modified procedure described in Lebofsky et al. (Lebofsky, Heilig et al. 2006). Briefly, HSV-1 particles were resuspended in 1X PBS at a concentration of 5 ⁇ 10 6 viral particles/mL, and mixed thoroughly at a 1:1 ratio with a 1.2% w/v solution of low-melting point agarose (Nusieve GTG, ref. 50081, Cambrex) prepared in PBS, at 50 °C. 90 ⁇ L of the viral particles /agarose mix was poured in a plug-forming well (BioRad, ref.
  • Embedded viral particles were lysed in 0.1% SDS - 0.5M EDTA (pH 8.0) solution at 50°C for 30 minutes. After three washing steps in 0.5M EDTA (pH 8.0) buffer of 10 minutes at room temperature, plugs were digested by overnight incubation at 50°C with 2 mg/mL Proteinase K (Eurobio code GEXPRK01, France) in 250 ⁇ L digestion buffer (0.5M EDTA, pH 8.0). The use of 0.1% SDS instead of Sarkosyl was very productive and allows a very high quality of extracted viral DAN to be collected.
  • HSV-1 DNA from infected cells culture was performed as previously described (Schurra and Bensimon 2009). Briefly, infected cells were pelleted by centrifugation at 5000g for 5 minutes, resuspended at a concentration of 2 ⁇ 10 6 cells/mL in 1X PBS buffer and mixed thoroughly at a 1:1 ratio with a 1.2% w/v solution of low-melting point agarose (Nusieve GTG, ref. 50081, Cambrex) prepared in 1 X PBS at 50°C. 90 ⁇ L of the cell / agarose mix was poured in a plug-forming well (BioRad, ref. 170-3713) and left to cool down at least 30 min at 4 °C.
  • Lysis of cells in the blocks was performed as previously described (Schurra and Bensimon 2009). Briefly, Agarose plugs were incubated overnight at 50 °C in 250 ⁇ L of a 0.5M EDTA (pH 8), 1 % Sarkosyl, 250 ⁇ g/mL proteinase K (Eurobio, code : GEXPRK01, France) solution, then washed twice in a Tris 10mM, EDTA 1 mM solution for 30 in at room temperature.
  • HSV-1 strain Sc16 infected mouse cornea was collected at a final stage of infection, and kept in Corneamax® (Eurobio code EYEMAX00, France) medium. After rinsing three times during 15 minutes at room temperature with 1X PBS solution, the entire cornea was cut into small pieces. Tissue lysis was carried out for up to 16h at 37°C in 0.3 mg/mL Collagenase type A (Roche, code 10 103 578 001), and 0.8 mg/mL GIBCOTM Dispase (Invitrogen, France, code 17105-041), both prepared in GIBCOTM 1X Hanks' Balanced Salt Solution HBSS buffer (Invitrogen, France, code 14060040).
  • Lysates were pelleted by centrifugation at 5000g for 10 minutes, resuspended at a concentration of 1•10 6 to 2•10 6 cells/mL in 1X PBS buffer and mixed thoroughly at a 1:1 ratio with a 1.2% w/v solution of low-melting point agarose (Nusieve GTG, ref. 50081, Cambrex) prepared in 1 X PBS at 50°C. 90 ⁇ L of the cell / agarose mix was poured in a plug-forming well (BioRad, ref. 170-3713) and left to cool down at least 30 min at 4 °C.
  • a plug-forming well BioRad, ref. 170-3713
  • Lysis of cells in the blocks was performed as previously described (Schurra and Bensimon 2009). Briefly, Agarose plugs were incubated overnight at 50 °C in 250 ⁇ L of a 0.5M EDTA (pH 8), 1 % Sarkosyl, 250 ⁇ g/mL proteinase K (Eurobio, code : GEXPRK01, France) solution, then washed twice in a Tris 10mM, EDTA 1 mM solution for 30 in at room temperature.
  • Plugs of embedded DNA from viral particles or corneas were treated for combing DNA as previously described (Schurra and Bensimon 2009). Briefly, plugs were melted at 68 °C in a MES 0.5 M (pH 5.5) solution for 20 min, and 1.5 units of beta-agarase (New England Biolabs, ref. M0392S, MA, USA) was added and left to incubate for up to 16h at 42° C. The DNA solution was then poured in a Teflon reservoir and Molecular Combing was performed using the Molecular Combing System (Genomic Vision S.A., Paris, France) and Molecular Combing coverslips (20 mm x 20 mm, Genomic Vision S.A., Paris, France). The combed surfaces were dried for 4 hours at 60 °C.
  • Probe size ranges from 1110 to 9325 bp in this example.
  • the HSV-1 specific probes were produced by either SacI or BspEI (New England Biolabs Inc., Beverly, MA, USA code R0156L and R0156L, respectively) enzymatic digestion of the HSV-1 scl6 strain obtained from the CNRS (Prof. Marc Labetoulle, dividend de virology molé Diagram et structurale, UMR CNRS 2472-INRA 1157, Gif-sur-Yvette, France) or by long-range PCR using LR Taq DNA polymerase (Roche, kit code: 11681842001) using the primers listed in table B and the DNA from HSV-1 sc16 as template DNA.
  • Sac I and BspE I HSV-1 fragments were ligated in SacI and XmaI-digested pNEB193 plasmid (New England Biolabs Inc., Beverly, MA, USA, code N3051S), respectively.
  • PCR products were ligated in the pCR®2.1 vector using the TOPO® TA cloning Kit (Invitrogen, France, code K455040). The two extremities of each probe were sequenced for verification purpose.
  • the apparent H1 (21 kb), H2 (56 kb), H3 (45 kb), H4 (13 kb), H5 (7.5 kb) and H6 (6.5 kb) probes are mixes of several adjacent or overlapping probes listed in table A.
  • the apparent H2 probe was split in two probes H2A (31 kb) and H2B (25 kb).
  • the labelling of the probes was performed using conventional random priming protocols.
  • biotin-11-dCTP labelling the BioPrime® DNA kit (Invitrogen, code: 18094-011, CA, USA) was used according to the manufacturer's instruction, except the labelling reaction was allowed to proceed overnight.
  • 11-digoxygenin-dUTP and Alexa488-7-OBEA-dCTP the dNTP mix from the kit was replaced by the mix specified in table C. 200 ng of each plasmid was labelled in separate reactions.
  • H1, H3, H5 probes were labelled with 11-digoxygenin-dUTP while H2, H4 and H6 probes were labelled with biotin-11-dCTP.
  • H1, H2B and H3 probes were labelled with biotin-11-dCTP, H2A and H5 probes with 11-digoxygenin-dUTP and H4, H6 with Alexa 488-7-OBEA-dCTP.
  • the reaction products were visualized on an agarose gel to verify the synthesis of DNA.
  • the probe solution and probes were heat-denatured together on the Hybridizer (Dako, ref. S2451) at 90 °C for 5 min and hybridization was left to proceed on the Hybridizer overnight at 37 °C.
  • Slides were washed 3 times in 50 % formamide, 2x SSC and 3 times in 2x SSC solutions, for 5 min at room temperature. Detection antibody layers and their respective dilution in Block-Aid are described in table D and E. For each layer, 20 ⁇ L of the antibody solution was added on the slide and covered with a combed coverslip and the slide was incubated in humid atmosphere at 37 °C for 20 min.
  • the slides were washed 3 times in a 2x SSC, 1 % Tween20 solution for 3 min at room temperature between each layer and after the last layer.
  • detection was carried out using a Texas Red coupled mouse anti digoxygenin (Jackson Immunoresearch, France) antibody in a 1:25 dilution for H1, H3, and H5 probes, and an Alexa488-coupled streptavidin antibody (Invitrogen, France) in a 1:25 dilution for H2, H4 and H6 probes as primary antibodies.
  • an Alexa594-coupled goat anti mouse (Invitrogen, France) diluted at 1:25 and a biotinylated goat antistreptavidin (Vector Laboratories, UK) diluted at 1:50 were used.
  • an additional detection layer was realized by using the same Alexa488 coupled-streptavidin used for the first layer at a 1:25 dilution.
  • detection was carried out using an AMCA-coupled mouse anti-digoxygenin (Jackson Immunoresearch, France) antibody in a 1:25 dilution for the H2A and H5 probes, an Alexa 594-coupled streptavidin antibody (Invitrogen, France) in a 1:25 dilution for the H1, H2B and H3 and a rabbit anti Alexa 488 in a 1:25 dilution as primary antibodies.
  • AMCA-coupled mouse anti-digoxygenin Jackson Immunoresearch, France
  • Alexa 594-coupled streptavidin antibody Invitrogen, France
  • H1, H2B and H3 Alexa 594-coupled streptavidin antibody
  • an Alexa 594-coupled goat anti mouse (Invitrogen, France) diluted at 1:25, a biotinylated goat anti-streptavidin (Vector Laboratories, UK) diluted at 1:50 and an Alexa 488-coupled goat anti-mouse diluted at 1:25 were used.
  • an additional detection layer was realized by using the same Alexa 594 coupled-streptavidin used for the first layer at a 1:25 dilution and an Alexa350 coupled goat anti rat diluted at 1:25, respectively.
  • all glass cover slips were dehydrated in ethanol and air dried.
  • cover slips were mounted with 20 ⁇ L of a Prolong (Invitrogen, France ref P36930)-YOYO-1 iodide (Molecular Probes, code Y3601) mixture (1/1000 v/v) and scanned with inverted automated epifluorescence microscope, equipped with a 40X objective (ImageXpress Micro, Molecular Devices, USA). Length of the YOYO-1-stained DNA fibers were measured and converted to kb using an extension factor of 2 kb/ ⁇ m (Schurra and Bensimon 2009), with an internal software GVlab 04.2.1 (Genomic Vision S.A., Paris, France).
  • hybridized-combed DNA from viral particles or cornea preparation were scanned without any mounting medium using an inverted automated epifluorescence microscope, equipped with a 40X objective (ImageXpress Micro, Molecular Devices, USA) and the signals can be detected visually or automatically by an in house software (Gvlab 0.4.2).
  • FISH signals composed of a continuous signal of Texas Red/ Alexa 594-fluorescence for H1, H3, H5, and Alexa 488-flurorescence for H2, H4 and H6, and signals composed of a continuous signal corresponding to one of the pattern described below were considered:
  • FISH signals selected were then classified depending of the pattern of the continuous FISH signals analysed:
  • hybridized-combed DNA from infected cells preparation were scanned without any mounting medium using an inverted automated epifluorescence microscope, equipped with a 40X objective (ImageXpress Micro, Molecular Devices, USA) and the signals can be detected visually on an in house software (Gvlab 0.4.2). All signals composed of a continuous signal of Alexa 488-fluorescence for H4 and H6 were selected. Signals corresponding to one of the pattern below were classified as canonical structure:
  • DNA molecules are sheared at random location due to uncontrolled manipulation forces resulting in high variability in the size of DNA prepared. It has been showed that high molecular weight DNA can be stretched on by Molecular Combing using a glass coverslip when it is deproteinised in a molten agarose plug (Lebofsky and Bensimon 2003). Thus, the analyzed DNA molecules are of variable length, with an average of about 300 kb, the longest molecules reaching several megabases.
  • the median size of DNA fibers is 36 kb with 1.2 % of fiber longer than 140 kb when extraction has been performed with the standard phenol:chloroform method.
  • the median size of HSV-1 DNA fiber is 84 kb with 2.5% of fiber longer than 140 kb when the extraction of DNA from agarose plug-embedded viral particles using our alternative protocol has been realized.
  • the proportion of long molecules is low, there are a lot of long combed DNA molecules available for analysis since there are several ten thousand fibers combed on a glass coverslip.
  • the inventors applied Molecular Combing to uniformly stretch the HSV-1 DNA extracted from viral particles and infected cells and hybridized the resulting combed HSV-1 DNA with labeled adjacent and overlapping HSV-1-specific DNA probes ( Fig. 1 ; H1, H3, H5: red Texas Red/Alexa 594-fluorescence; H2, H4, H6: green Alexa 488-fluorescence) to determine the structure of the HSV-1 genome ( Fig. 3A ).
  • Immunofluorescence microscopy ( Fig. 3B .) exhibited 405 multicolor linear patterns for each production of HSV-1 KOS strain viral particles produced from COS7, Vero and Neuro2 A cell lines that fulfilled the criteria for evaluation (see “Examples" section).
  • the inventors have found that the Molecular Combing techniques as described herein are powerful methods for analysis of the structure of the HSV-1 genome DNA at the level of the unique molecule and to quantify its distribution in a biological sample.
  • the human cornea is a solid tissue with a complex structure composed of 5 layers: the corneal epithelium, the collagen-rich Bowman's membrane, the corneal stroma which consisting of regularly-arranged collagen fibers along with sparsely distributed interconnected keratocytes), the acellular Descemet's membrane and, the corneal endothelium.
  • Figure 4A and B shows an example of results obtained with both mouse and rabbit cornea that were digested with 0.3 mg/ml collagenase A and 0.8 mg/ml Dispase for 16h.
  • As for standard extraction for Molecular Combing genomic DNA is broken at random locations.
  • the analyzed genomic DNA molecules are of variable length, with an average of about 200 kb, the longest molecules above 1 Mb (megabases) for both type of cornea.
  • the size of DNA fiber extracted from cornea is slightly inferior to the typical size that can be obtained from isolated cells (an average of about 300 kb with the longest molecules reaching several megabases).
  • the inventors therefore adapted and applied Molecular Combing on DNA extracted from HSV-1 infected mouse cornea and hybridized with the HSV-1 specific probes as described above. As shown in Fig. 5 , this enabled detection all the types of isomers of HSV-1 genome in mouse infected cornea.
  • the Molecular Combing procedures of the invention allow the detection of concatemers ( Fig. 6 ) indicating that the virus is actively replicating in the cornea of the analyzed sample.
  • the inventors detected non-canonical structure of the HSV-1 genome ( Fig. 7A ) that probably arises from recombination during the replication of the virus in infected mouse cornea extract and in infected cells extracts.
  • H1, H2A, H3 red Alexa 594-fluorescence that appears in grey
  • H2B, H5 blue AMCA/ Alexa 350-fluorescence that appears in white
  • H4, H6 green Alexa 488-fluorescence that appears in black
  • composition of the 2 or 3 layers for the detection of probes by fluorescence Composition of the 2 or 3 layers for the detection of probes by fluorescence.
  • the dilution for each detection agent is indicated in brackets.
  • the abbreviations refer to table D.
  • ACH-2 cell lines (Clouse, Powell et al. 1989) were cultivated according to the authors' instructions. DNA was extracted as described in (Schurra and Bensimon 2009). Briefly, cells were resuspended in 1 X PBS at a concentration of 10 7 cells / mL mixed thoroughly at a 1:1 ratio with a 1.2% w/v solution of low-melting point agarose (Nusieve GTG, ref. 50081, Cambrex) prepared in 1 X PBS at 50°C. 90 ⁇ L of the cell / agarose mix was poured in a plug-forming well (BioRad, ref. 170-3713) and left to cool down at least 30 min at 4 °C.
  • Agarose plugs were incubated overnight at 50 °C in 250 ⁇ L of a 0.5M EDTA (pH 8), 1 % Sarkosyl, 250 ⁇ g/mL proteinase K (Eurobio, code : GEXPRK01, France) solution, then washed twice in a Tris 10mM, EDTA 1 mM solution for 30 in at room temperature.
  • a 0.5M EDTA pH 8
  • 1 % Sarkosyl 250 ⁇ g/mL proteinase K (Eurobio, code : GEXPRK01, France) solution
  • Tris 10mM, EDTA 1 mM solution for 30 in at room temperature.
  • Plugs of embedded DNA from ACH-2 cells were treated for combing DNA as previously described (Schurra and Bensimon 2009). Briefly, plugs were melted at 68 °C in a MES 0.5 M (pH 5.5) solution for 20 min, and 1.5 units of beta-agarase (New England Biolabs, ref. M0392S, MA, USA) was added and left to incubate for up to 16h at 42° C. The DNA solution was then poured in a Teflon reservoir and Molecular Combing was performed using the Molecular Combing System (Genomic Vision S.A., Paris, France) and Molecular Combing coverslips (20 mm x 20 mm, Genomic Vision S.A., Paris, France). The combed surfaces were dried for 4 hours at 60 °C.
  • Probe size ranges from 2927 to 3749 bp in this example.
  • the HIV specific probes were produced by long-range PCR using LR Taq DNA polymerase (Roche, kit code: 11681842001) using the primers listed in table G and the DNA from HIV pNL4-3 as template DNA. PCR products were ligated in the pCR®2.1 vector using the TOPO® TA cloning Kit (Invitrogen, France, code K455040). The two extremities of each probe were sequenced for verification purpose.
  • the dNTP mix from the kit was replaced by the mix specified in table C. 200 ng of each plasmid/ fosmid was labelled in separate reactions. For entire HIV-1 detection, HIV-1 was labelled with digoxygenin-11-dUTP while fosmids were labelled with biotin-11-dCTP. The reaction products were visualized on an agarose gel to verify the synthesis of DNA.
  • the probe solution and probes were heat-denatured together on the Hybridizer (Dako, ref. S2451) at 90 °C for 5 min and hybridization was left to proceed on the Hybridizer overnight at 37 °C.
  • Slides were washed 3 times in 50 % formamide, 2x SSC and 3 times in 2x SSC solutions, for 5 min at room temperature. Detection antibody layers and their respective dilution in Block-Aid are described in table D and E. For each layer, 20 ⁇ L of the antibody solution was added on the slide and covered with a combed coverslip and the slide was incubated in humid atmosphere at 37 °C for 20 min. The slides were washed 3 times in a 2x SSC, 1 % Tween20 solution for 3 min at room temperature between each layer and after the last layer.
  • Detection of entire HIV-1 was carried out using a Texas Red coupled mouse anti-digoxygenin (Jackson Immunoresearch, France) antibody in a 1:25 dilution for HIV-1 probes, and an Alexa488-coupled streptavidin antibody (Invitrogen, France) in a 1:25 dilution for fosmids as primary antibodies.
  • an Alexa594-coupled goat anti mouse Invitrogen, France
  • a biotinylated goat antistreptavidin Vector Laboratories, UK
  • an additional detection layer was realized by using the same Alexa488 coupled-streptavidin used for the first layer at a 1:25 dilution. After the last washing steps, all glass cover slips were dehydrated in ethanol and air dried.
  • Hybridized-combed DNA from ACH-2 cells preparation were scanned without any mounting medium using an inverted automated epifluorescence microscope, equipped with a 40X objective (ImageXpress Micro, Molecular Devices, USA) and the signals can be detected visually or automatically by an in house software (Gvlab 0.4.2):
  • the inventors have applied Molecular Combing to detect complete HIV-1 integrated provirus in ACH-2 cell lines (Clouse, Powell et al. 1989), which contain a unique integrated form of HIV-1 in its genome in NT5C3 (Cytosolic 5'-nucleotidase III) gene on 7p14.3 (Ishida, Hamano et al. 2006). Labeled fosmids flanking the insertion site (G248P87988G9 and G248P86255A8) were hybridized on combed ACH-2 DNA simultaneously than labelled HIV-1 probes.
  • a mean size of 10.2 kb +/-0.8kb was obtained from measurement of 124 HIV-1 FISH signals flanking by one or both fosmids, corresponding to the expected size of HIV-1 (9,7 kb) ( Figure 8A , HIV probes: red Texas Red/Alexa 594-fluorescence, Fosmids: green Alexa 488-fluorescence). Normal alleles of NT5C3 gene are detected and measurement of the gap length between fosmids G248P87988G9 and G248P86255A8 FISH signal leads to a mean size of 32.8 ⁇ 1.8 kb, lightly superior to the expected size of 31 kb, according to Human Mar. 2006 Assembly (NCBI Build 36.1).
  • Primers sequences used for the synthesis of probes by long-range PCR An extract of DNA from HIV-1 pNL-4 is used as template.
  • probes are designed to detect the presence of a viral oncogene. Probes are designed to complement 80-100% of the active viral oncogene of interest and Molecular Combing is performed. The results indicate the presence of an active oncogene in a subject, leading to diagnosis and therapeutic intervention.
  • probes are designed to detect the presence of different arrangements of infectious viral DNA.
  • Different sets of probes tagged with different haptens recognized by different colored fluorescent probes are designed to complement 80-100% of the active viral oncogene of interest and Molecular Combing is performed.
  • Variations in the arrangement of viral genes in a subject's cells, tissue or biological fluid are used to diagnose or prognose the risks of progression of the viral disease or disease associated with rearrangement of the viral genome, such as the risk of or induction of a tumorigenic properties by conversion of proto-oncogenes into oncogenes.
  • probes are specially designed to complement 80-100% of an integrated therapeutic adenovirus vector or the transgenetic sequence(s) it carries.
  • Molecular Combing is performed using the specially designed probes to detect the presence of transgenic material integrated into a host chromosome and whether it is arranged in form that can actively express the transgene(s).
  • the quantity of transgene(s) in the subject is followed longitudinally and a determination is made when and whether to re-administer the therapeutic adenovirus vector.

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